1. Field of the Invention
The present invention is directed to the immortalizing E6 and E7 genes of human papilloma viruses, such as virus types 16, 18, 31, 33 and 35 and transfection of these genes into target cells. In vivo expression of E6/E7 genes immortalizes the target cell which retains its phenotypic characteristics.
2. Discussion of the Background
More than 50 different types of human papillomariruses (HPVs) have now been isolated from a variety of squamous epithelial lesions, and approximately 18 of them have been associated with anogenital tract lesions. Some of these, such as HPV type 6 (HPV-5) and HPV-11, are generally associated with benign proliferative lesions, including condyloma acuminata, which only infrequently progress to cancers. Others, such as HPV-16, HPV-18, HPV-31, HPV-33, and HPV-35, are associated with genital tract lesions, which are at risk for malignant progression, and with genital tract cancers (1).
HPV-16 or HPV-18 DNA has been found integrated in a high percentage of cervical carcinomas and in cell lines derived from these cancers (2, 3, 4). This is in contrast with the premalignant dysplastic lesions associated with HPV-16 and HPV-18, in which the viral DNA is usually found in an extrachromosomal state (5). In several cases in which the number of integrated viral genomes was low enough to permit a detailed analysis, the integration pattern revealed remarkable specificity with respect to the circular viral genome. Integration occurs in the E1-E2 region (6, 7, 8), disrupting the E2 viral transcriptional regulatory circuitry. The E2 open reading frame (ORF), as originally demonstrated with the bovine papillomavirus type 1, encodes both positive- and negative-acting transcriptional regulatory factors (9, 10). For HPV-16 and HPV-18, E2 appears to act principally as a repressor of the promoter from which the E6 and E7 genes are transcribed (11, 12). The HPV genomes in cervical carcinomas and in derived cell lines are transcriptionally active, and the patterns of viral mRNA species are specific, with regular expression of the E6 and E7 ORRFs (6, 13, 14).
The E7 ORF of HPV-16 encodes a 21-kilodalton phosphoprotein (14), and the E7 genes of HPV-16 and HPV-18 are sufficient for focus formation of established rodent fibroblasts such as NIH 3T3 cells (15, 16, 17, 18, 19). The E7 protein is functionally and structurally related to the adenovirus E1A proteins (AdE1A); it can transactivate the AdE2 promoter and can cooperate with an activated ras oncogene to transform primary rat cells (16, 20). The amino-terminal 38 amino acids of E7 are strikingly similar to portions of conserved domain 1 (amino acids 37 to 49) and domain 2 (amino acids 116 to 137) of the AdE1A proteins (16) as well as to portions of the large tumor antigens (T) of papovaviruses. The AdE1A, simian virus 40 (SV40) T, and HPV-16 E7 proteins form specific complexes with the product of the retinoblastoma tumor suppressor gene (p105-RB) (21, 22, 23), and complex formation with p105-RB is mediated through these conserved sequences for AdE1A and SV40 T (21, 22) as well as for HPV-16 E7. The transforming potential of the E6 gene has been less well defined. In NIH 3T3 fibroblasts, it may contribute to characteristics of the transformed phenotype such as anchorage independence (25) or tumorigenicity in nude mice (26). In human cells, E6 appears to cooperate with the E7 oncopprotein in mediating-cellular immortalization. Recently, it has been demonstrated that E6 binds to, and mediates the degradation of, the cellular tumor suppressor protein p53. It has been shown recently that both the E6 and E7 RFs are necessary for the extension of the life span of human diploid fibroblasts (34). Mutation studies of the early HPV-16 genes that directly participate in the in vitro transformation of primary human keratinocytes has shown that both the full-length E6 and E7 genes are required for induction of keratinocyte immortalization and resistance to terminal differentiation (35). Keratinocyte transformation with HPV-18 DNA requires only the HPV-18 regulatory region and the E6/E7 genes which induce two progressive steps in cellular transformation (36).
A quantitative keratinocyte assay has been used to demonstrate that the HPV genes can alter the response of human keratinocytes to inducers of terminal differentiation. HPV-16 and HPV-18 DNA can immortalize human keratinocytes in vitro. These immortalized cell lines exhibit altered characteristics of cellular proliferation and differentiation but are not tumorigenic in nude mice. They contain integrated copies of HPV DNA and express viral mRNAs and proteins (27, 28, 29, 30, 31, 32). While the above in-vitro assays rely on the growth of keratinocytes on plastic substrate and can be criticized for its lack of physiologic relevance, other in vitro assays which more closely mimic the in vivo conditions for squamous cell growth have demonstrated similar results. Thus, when human keratinocytes are grown by the collagen raft cell culture technique, they display an enhanced cellular differentiation and stratification which is very similar to, but not identical with, that observed of keratinocytes in vitro. This technique has been successfully applied to the study of HPV gene effects on cellular differentiation and several laboratories have demonstrated that the E6/E7 genes produce altered cellular differentiation reminiscent to that observed in cervical dysplasia (33). Thus, all experimental data to date suggests that the expression of the E6/E7 genes results in a poorly differentiated phenotype when assayed in vitro.
(In a recent study, HPV-16 immortalized human keratinocytes were subcutaneously injected into nude mice. Although the immortalized cells retain the ability for differentiation after injection, the injected cells were immunoisolated to form encapsulated cysts).
The study of normal cell growth and differentiation would be greatly augmented by the development of an efficient method for obtaining human immortalized cell lines which would retain their ability to differentiate and respond to external regulatory signals. One critical research area which would greatly benefit from such an approach would be the study of cystic fibrosis (CF). Not only would CF cell lines permit the analysis of the altered ion permeability properties of these cells and their alteration by pharmacologic agents, but they would also serve as a substrate for future gene therapy experiments. In an attempt to generate such cell lines, the SV40 large T antigen has been used to immortalize CF cells. Unfortunately, the derived cell lines lose many of their differentiated properties and are inadequate for biochemical, physiological, and molecular analysis.
A need continues to exist for a means of immortalizing human cells in a non-tumorigenic manner such that the immortalized cells retain their differentiated phenotypic properties. A broadly applicable means for producing non-tumorigenic immortalized cell lines would facilitate research on gene products of particular cells, provide cell lines capable of producing large quantities of gene products without the need to replace senescent cell lines; provide immortalized cell lines for use in direct gene therapy applications and also provide a means of immortalizing cell lines containing exogenous genes or genes which have been subjected to site-specific mutation to correct abnormalities in gene expression.